Electrophoretic separations of bio-molecules are critically important in modern biology and biotechnology, comprising an important component of such techniques as DNA sequencing, protein molecular weight determination, genetic mapping, and the like. A particularly preferred electrophoresis format is capillary electrophoresis (CE), where the electrophoresis is performed in a channel, such as a capillary tube or a groove in a microfabricated chip, wafer or plate, having a small internal diameter. Capillary electrophoresis results in enhanced separation performance over traditional slab-based formats because the superior ability of the narrow-bore capillary to tolerate resistive heating allows for high electrical fields to be employed thereby resulting in fast separations in which sample diffusion is minimized.
In traditional CE systems, detection of a sample subsequent to separation is performed during electrophoresis while the sample is still located inside the channel (referred to as “on-channel” detection). Thus, in a common capillary tube arrangement, any excitation light required to excite the sample and any emission light coming from the sample must be transmitted through the wall of the capillary tube. A drawback of this approach is that the fused silica capillary tubes often used in CE have numerous surfaces to reflect or scatter light. Problems associated with light scattering are particularly problematic when it is desired to detect fluorescence from samples located in a plurality of closely-spaced capillary tubes by fluorescence because the scattered emmission light from one capillary tube can interfere with the detection of samples in neighboring capillary tubes.
One approach to solving the problem of on-channel detection has been to detect a sample after the sample emerges from the capillary (referred to as “off-channel” detection). In one type of arrangement, such off-channel detection takes place in a detection cell positioned downstream of the capillary tube outlets. Typically, the detection cell is configured to exhibit superior optical characteristics, e.g., a flat quartz chamber. In one class of these systems, a “sheath flow” of liquid is used to transport the sample from the outlet of the CE capillary tube to a detection zone at which detection of the sample takes place (Takahashi; Dovichi). A drawback of sheath flow systems is that, in order to avoid distortion of a sample zone in the detection cell, precise control of the flow rate of the sheath flow liquid is required. A second drawback of sheath flow systems is that the pressure used to drive the flow of the sheath flow liquid can cause back flow of the separation medium in the separation capillary tube, thereby negatively impacting resolution.
In another class of off-channel detection systems, a sample zone is transported from the outlet of a CE capillary tube to a detection zone located in a detection cell by electrophoresis under the influence of the same voltage difference used to conduct the electrophoretic separation (Takahashi). However, because of the larger cross-sectional area within the detection cell as compared to the lumen of the capillary tube, the electric field diverges at the capillary tube outlet causing a distortion of the sample zone as it enters and traverses the detection zone. Unchecked, such distortion can result in a severe loss of spatial resolution between adjacent sample zones exiting a single capillary tube and/or between sample zones exiting adjacent capillary tubes. This loss of spatial resolution tends to reduce the detectability of neighboring sample zones.